including ceramide, sphingosine (SPH), and SPH-1-phosphate (S1P), have been shown to be involved in different cell functions such as proliferation, differentiation, growth arrest, and apoptosis ( 2 ). Especially the counterregulatory functions of ceramide and S1P, resembling the sphingolipid rheostat, indicate that not only a single metabolite concentration but rather the relative levels of these lipids are important to determine the cell fate ( 2-5 ). Sphingolipids are associated with several diseases such as cancer, obesity, and atherosclerosis ( 1, 2, 6-9 ). Structural diversity and inter-conversion of these sphingolipid metabolites represent technical challenges. Nevertheless, to understand the differential role of sphingolipids in a regulatory network, it is imperative to use specifi c and quantitative methods.During the last decade, LC-MS/MS has become a powerful tool for sphingolipid analysis ( 10-21 ). However, either these methods do not cover a broad spectrum of sphingolipid metabolites or they show disadvantages like laborious sample preparation, time-consuming LC-separation, or separation of analytes and internal standards (ISs).Therefore, we applied, as previously described for lysophosphatidic acid and sphingoid base phosphates, hydrophilic interaction chromatography (HILIC) coupled to MS ( 18 ) to achieve coelution of sphingolipid species and their ISs. We present a fast and simple LC-MS/MS method for the quantifi cation of hexosylceramide (HexCer), lactosylceramide (LacCer), sphingosine (SPH), sphinganine (SPA), phyto-SPH (PhytoSPH), di-and trimethyl-SPH (Di-; TrimetSPH), sphingosylphosphorylcholine (SPC), ceramide-1-phosphate (Cer1P), and dihydroceramide-1-phosphate (dhCer1P). This method was validated and apAbstract Sphingolipids comprise a highly diverse and complex class of molecules that serve not only as structural components of membranes but also as signaling molecules. To understand the differential role of sphingolipids in a regulatory network, it is important to use specifi c and quantitative methods. We developed a novel LC-MS/MS method for the rapid, simultaneous quantifi cation of sphingolipid metabolites, including sphingosine, sphinganine, phyto-sphingosine, di-and trimethyl-sphingosine, sphingosylphosphorylcholine, hexosylceramide, lactosylceramide, ceramide-1-phosphate, and dihydroceramide-1-phosphate. Appropriate internal standards (ISs) were added prior to lipid extraction. In contrast to most published methods based on reversed phase chromatography, we used hydrophilic interaction liquid chromatography and achieved good peak shapes, a short analysis time of 4.5 min, and, most importantly , coelution of analytes and their respective ISs. To avoid an overestimation of species concentrations, peak areas were corrected regarding isotopic overlap where necessary. Quantifi cation was achieved by standard addition of naturally occurring sphingolipid species to the sample matrix. The method showed excellent precision, accuracy, detection limits, and robustness. As an example, sphingol...
Microglial cells, in contrast to other central nervous system cell types such as neurons and macroglia, are of myeloid origin. They constitute the immune cells of the brain and are involved in neuroinflammatory and neurodegenerative processes. Moreover, diseases of the central nervous system with an inflammatory component are characterized by the migration of bone marrow-derived monocytes into the brain where they differentiate into microglia, the "tissue macrophages" of the nervous system, bearing a therapeutic potential for certain diseases by transplantation of bone marrow-derived hematopoietic stem and progenitor cells. Due to their common origin, microglial cells and monocytes/macrophages share expression of many surface receptors and signalling proteins. Moreover, there is overlap in the expression of many genes related to Alzheimer s disease. Activation of resident and blood-derived microglia in diseases of the central nervous system can be both beneficial, e.g. by degradation of protein aggregates, and detrimental, e.g. by secretion of neurotoxic factors. This review summarizes the current knowledge about the role of microglia in neurodegenerative diseases with a focus on Alzheimer s disease. Moreover, we present data how neuroinflammation is reflected by cellular changes in peripheral blood enabling the use of blood monocytes/macrophages for diagnosis, therapeutic target finding and outcome monitoring of neurodegenerative disorders. In summary, blood monocytes as microglia orthologues are an important model system to study the role of microglia in the pathogenesis of neurodegenerative diseases. They are suitable biomarker targets for diagnosis and prognosis and maybe also therapy of central nervous system disease.
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